A Phylogenetic Analysis of Greek Isolates of Aspergillus Species Based on Morphology and Nuclear and Mitochondrial Gene Sequences

Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 260395, 18 pages http://dx.doi.org/10.1155/2013/260395

Research Article A Phylogenetic Analysis of Greek Isolates of Aspergillus Species Based on Morphology and Nuclear and Mitochondrial Gene Sequences

Antonios Krimitzas,1 Ioanna Pyrri,2 Vassili N. Kouvelis,1 Evangelia Kapsanaki-Gotsi,2 and Milton A. Typas1

1 Department of Genetics and Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistemiopolis, 15701 Athens, Greece 2 Department of Ecology and Systematics, Faculty of Biology, National and Kapodistrian University of Athens, Panepistemiopolis, 15784 Athens, Greece

Correspondence should be addressed to Milton A. Typas; [email protected]

Received 13 March 2013; Accepted 9 April 2013

Academic Editor: George Tsiamis

Copyright © 2013 Antonios Krimitzas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Aspergillus species originating from Greece were examined by morphological and molecular criteria to explore the diversity of this genus. The phylogenetic relationships of these species were determined using sequences from the ITS and IGS region of the nuclear rRNA gene complex, two nuclear genes (𝛽-tubulin (benA) and RNA polymerase II second largest subunit (rpb2)) and two mitochondrial genes (small rRNA subunit (rns) and cytochrome oxidase subunit I (cox1)) and, where available, related sequences from databases. The morphological characters of the anamorphs and teleomorphs, and the single gene phylogenetic trees, differentiated and placed the species examined in the well-supported sections of Aenei, Aspergillus, Bispori, Candidi, Circumdati, Clavati, Cremei, Flavi, Flavipedes, Fumigati, Nidulantes, Nigri, Restricti, Terrei, Usti,andZonati, with few uncertainties. The combined use of the three commonly employed nuclear genes (benA, rpb2, and ITS), the IGS region, and two less often used mitochondrial gene sequences (rns and cox1) as a single unit resolved several taxonomic ambiguities. A phylogenetic tree was inferred using Neighbour-Joining, Maximum Parsimony, and Bayesian methods. The strains examined formed seven well- supported clades within the genus Aspergillus. Altogether, the concatenated nuclear and mitochondrial sequences offer additional tools for an improved understanding of phylogenetic relationships within this genus.

1. Introduction worldwide distribution necessitates the detailed taxonomic and phylogenetic study of Aspergillus species in order to The genus Aspergillus is one of the most common and provide more appropriate tools for their accurate and fast important genera of microfungi. Several species of the genus identification. proved very useful cell factories for biotechnology purposes The first most comprehensive taxonomic treatise of the since they produce organic acids, extracellular enzymes, genuswasbasedonmorphologicalcriteriaandincluded nutraceuticals (isoprenoids), and aroma compounds and 150 taxa that were separated in “groups” [7]. These clusters are capable of fermenting various substrates under extreme of species (“groups”) were later revised to 18 “sections” in cultivation conditions, usually intolerable to bacteria and 6 subgenera, in order to receive nomenclatural status [8]. yeasts [1–4]. Furthermore, the genus contains a number of In followup, a list of 182 species valid names was compiled species that produce toxic metabolites, act as opportunistic [9], and expanded to ∼250 species [3], and it is expected to pathogens to humans, or decompose a wide variety of increase further as new species are discovered and speciation substrates [5, 6]. Their high diversity combined with the concepts are refined [10]. 2 BioMed Research International

During the last decade, a revision of the genus Aspergillus The strains of Aspergillus were three-point inoculated is in progress, as a result of molecular analyses which have inselectivenutrientmediaandincubatedonCzapekYeast ∘ ∘ offered a better insight into taxonomic and phylogenetic rela- Autolysate Agar (CYA) at 25 Cand37C, on Malt Extract ∘ ∘ tions [10–13]. The revision is based on polyphasic approaches Agar (MEA) at 25 C,andonG25Nat25C[29]. The that include molecular data, as well as morphological, phys- macromorphology of the colonies on each of the media, as iological and ecological characteristics. Although no single well as the micromorphology on MEA, were studied after 7 method worked flawlessly in recognising species, molecular days of incubation. Microscopic examination was performed data have largely supported previously inferred relationships by teasing apart the sample in a drop of 70% ethanol on a glass that were based on the other characters [3]. Single locus DNA slide, a coverslip was placed on it, and it was observed under sequence studies were a common practice in fungi in the past, a Zeiss microscope in plain light or Differential Interference thus, large amounts of information have been accumulated Contrast. The photographs were taken with an Axiocam in databases for several parts of the nuclear ribosomal rRNA digital camera (Zeiss, Germany). When the evaluation of the repeat (in particular the ITS and 28S) [14–16] and genes like morphological data was completed, all strains were further the 𝛽-tubulin [17]. The molecular identification of Aspergillus examined with molecular markers and the data was used speciesiscurrentlybasedonsequencesfromgenes𝛽-tubulin, in phylogenetic analyses. Additional taxa were included to calmodulin, actin, and ITS [5, 12, 13, 18–22]. A phylogenetic represent most of the sections recognized in the genus study of Aspergillus species,basedonfoursinglenuclear Aspergillus. genes that were concatenated and used as one unit, attempted Mycelium preparation and fungal DNA extraction pro- to resolve the existing taxonomic ambiguities and has resulted cedures used are previously described [30]. PCR primers in a reevaluation of the genus Aspergillus [23, 24]. used in the amplification reactions for the ITS1-5.8S-ITS2 In other fungal genera, like Lecanicillium, Verticillium, region were 18S-ITS1 and 28S-ITS2 [27]andfortheIGS and Beauveria, the use of mitochondrial (mt) genes and region were CNL12 and CNS1 [31]. For the nuclear genes mt intergenic regions proved an extremely useful tool to andspecificallyfortheRNApolymeraseIIsecondlargest reveal species differences within a genus and even helped to subunit gene (rpb2), the primers used were RPB2-6F and resolve taxonomic ambiguities (e.g., the small rRNA subunit RPB2-7R [32]andforthe𝛽-tubulin gene (benA) the primers (rns), the NADH dehydrogenase subunits 1 (nad1) and 3 were bt2a and bt2b [33]. Primers for the amplification of mt (nad3), the mt intergenic domains nad3–atp9andtheatp6- genes were NMS1 and NMS2 for rns [34] and cox1F and cox1R rns)[25–27]. Similarly, other nuclear regions/genes like the for cox1[35]. The PCR amplicons were sequenced using a RNA polymerase II largest subunit gene (rpb1) and the IGS Thermo Sequenase Primer Cycle Sequencing kit (Amersham region of the rRNA repeat also proved to be excellent tools Biosciences, Amersham, UK), with a LICOR 4200 IR2 (LI- for discriminating species within Metarhizium, Verticillium COR, Lincoln NE, USA) automated sequencer. All fragments and Aspergillus [23, 27, 28]. were read in both directions and nucleotide sequences were In the present study the efficiency of combined nuclear submitted to GenBank database (Table 1). and mitochondrial gene sequence data was evaluated for Maximum parsimony (MP), Neighbour-Joining (NJ), the identification of species that are distributed in most of and Bayesian inference (BI) analyses were employed in order Aspergillus sections. In detail, the commonly used (ITS1-5.8S- to create the phylogenetic trees. MP, and NJ analyses of ∗ ITS2, RNA polymerase II second largest subunit gene (rpb2) nucleotide datasets were performed with PAUP 4.0b10 [36] and b-tubulin (benA)) and the less often exploited (IGS) using 1,000 and 10,000 replicates, respectively, with random nuclear regions were assessed along with two mitochondrial addition of taxa and tree-bisection reconnection branch genes, cytochrome oxidase subunit I (cox1) and small rRNA swapping. Reliability of nodes was assessed using 1,000 and subunit (rns), for the identification of Aspergillus species and 100 bootstrap iterations (for NJ and MP analyses, resp.) the evaluation of their phylogenetic affinities. The objective and for the NJ analyses the Kimura-2 parameter model was of this work is to test hypotheses based on morphological employed. The BI was performed with MrBayes v3.1 [37]. criteria and data obtained from a multilocus DNA sequence A gamma distribution model of site variation was used, analysis on the phylogeny within the genus Aspergillus. calculated with PAML [38]. For datasets ITS1-5.8S-ITS2, IGS, benA, rpb2, rns, cox1 and the concatenated data, alpha (𝛼) was 0.470, 0.630, 0.540, 0.400, 0.348, 0.527, and 0.403, 2. Materials and Methods respectively. Random starting trees were used and the burn- in period was 1,000,000 cycles, as this was found to be clearly Several strains of Aspergillus have been isolated from the sufficient for the likelihood and the model parameters to indoor air of a food industry in Greece, as well as from reach equilibrium. After the burn-in, 1,000 trees were sam- food samples. Additional strains of representative Aspergillus pled every 100 cycles during the sampling period (1,000,000 spp. were studied from the ATHUM Culture Collection cycles). Two independent MCMCMC searches were run for of Fungi in the University of Athens and ex-type cultures each dataset using different random starting points. The were obtained from the Agricultural Research Service ARS hypocrealean fungus Metarhizium anisopliae from the Class (NRRL) at Peoria, USA (kindly provided by Dr. S. Peterson). Sordariomycetes, a close relative to Eurotiomycetes, was used A total of thirty six strains belonging to thirty-one Aspergillus as outgroup in all datasets. When the species Coccidioides species were studied (Table 1). All strains are maintained in immitis from Onygenales, an order within Eurotiomycetes, the ATHUM Culture Collection of Fungi. was alternatively employed, the tree topologies from all BioMed Research International 3 rns 1 cox 2 rpb A ben GenBank accession number ITS IGS AF218207 AF218207 AY995134 DQ522453 AY884128 AY884128 sppusedinthisstudy. USA EF652141 EU982054 EF652123 EF652089 EU982135 EU982168 Aspergillus C USA EF661208 EU982052 EF661121 EF661077 EU982133 EU982166 ∘ Dalbergia sissoo Indoor air Greece EU982028 EU982062 EU982087 NT EU982142 EU982176 Forest soil Somalia EF652474 EU982060 EF652298 EF652210 EU982140 EU982174 Table 1: The strains of a b NRRL 10 Bat dung Mexico EF669928 NT EF669789 EF669716 EU982146 EU982181 NRRL 415 Food, fruit (apple) USA EF661400 EU982064 EF661337 EF661287 NT EU982178 NRRL 154 Clinical isolate from cloth England EF652042 EU982053 EF651880 EF651978 EU982134 EU982167 NRRL 5113 Forest soil Australia EF669713 EU982058 EF669686 EF669668 NT EU982172 NRRL 5176 Alkaline soil India EF652502 EU982059 EF652326 EF652238 EU982139 EU982173 NRRL 1787 Soil Panama EU021598 EU982061 EU014076 EF669620 EU982141 EU982175 NRRL 5012 Soil India EU021615 EU982048 EU021674 EU021653 NT NT NRRL 3693 Soil stored 10 years at 30 NRRL 4273 soil under NRRL 13001 Native mixed prairie soil USA EU014091 EU982049 EF669577 EF669619 EU982130 EU982163 NRRL 4769 ATHUM 5103 Dairy product Greece EU982027 EU982057 EU982086 EU982109 EU982138 EU982171 ATHUM 5013 Indoor air Greece EU982013 EU982038 EU982072 EU982097 EU982120 EU982153 ATHUM 5015 Indoor air Greece EU982011 EU982036 EU982070 EU982095 EU982118 EU982151 ATHUM 4761 Outdoor air Greece EU982024 EU982051 EU982083 EU982106 EU982132 EU982165 ATHUM 5019 Indoor air Greece EU982030 EU982065 EU982089 EU982112 EU982144 EU982179 ATHUM 2541 Culture contaminant Greece EU982032 NT EU982091 EU982114 EU982147 EU982182 ATHUM 5014 Indoor air Greece EU982029 EU982063 EU982088 EU982111 EU982143 EU982177 ATHUM 5033 Dairy product Greece EU982012 EU982037 EU982071 EU982096 EU982119 EU982152 ATHUM 2539 Outdoor air Greece EU982009 EU982034 EU982068 EU982092 EU982116 EU982149 ATHUM 5032 Dairy product Greece EU982014 EU982039 EU982073 EU982098 EU982121 EU982154 ATHUM 5037 Dairy product Greece EU982020 EU982045 EU982079 NT EU982127 EU982160 ATHUM 5022 Indoor air Greece EU982031 EU982066 EU982090 EU982113 EU982145 EU982180 ATHUM 5038 Dairy product Greece EU982021 EU982046 EU982080 NT EU982128 EU982161 ATHUM 5093 Dairy product Greece EU982025 EU982055 NT NT EU982136 EU982169 ATHUM 5029 Indoor air Greece EU982023 EU982050 EU982082 EU982105 EU982131 EU982164 ATHUM 5082 Indoor air Greece EU982017 EU982042 EU982076 NT EU982124 EU982157 ATHUM 4958 Outdoor air Greece EU982022 EU982047 EU982081 EU982104 EU982129 EU982162 ATHUM 5036 Dairy product Greece EU982015 EU982040 EU982074 EU982099 EU982122 EU982155 ATHUM 5030 Dairy product Greece EU982016 EU982041 EU982075 EU982100 EU982123 EU982156 ATHUM 5434 Dairy product Greece EU982019 EU982044 EU982078 EU982101 EU982126 EU982159 ATHUM 5097 Indoor air Greece EU982026 EU982056 EU982085 EU982108 EU982137 EU982170 ATHUM 5044 Dairy product Greece EU982008 EU982033 EU982067 EU982093 EU982115 EU982148 ATHUM 5028 FRR 4795 = ATHUM 5181 Unrecorded source Unknown EU982010 EU982035 EU982069 EU982094 EU982117 EU982150 FRR 0326 = ATHUM 5183 Stored pack of prunes Australia EU982018 EU982043 EU982077 NT EU982125 EU982158 T T 𝑢𝑠 T V T T T T T T T 𝑎𝑡𝑜𝑓𝑙𝑎 V NRRL: National Center For Agricultural Utilization Research. ATHUM: Culture Collection of Fungi, National and Kapodistrian University of Athens. 𝐴. 𝑐𝑙𝑎 SpeciesA. aculeatus Accession number Substrate Origin A. awamori 𝐴. 𝑏𝑖𝑠𝑝𝑜𝑟𝑢𝑠 A. fumigatus 𝐴. 𝑔𝑖𝑔𝑎𝑛𝑡𝑒𝑢𝑠 A. flavus E. variecolor E. rubrum A. sydowii A. terreus A. ustus A. versicolor Emericella nidulans Eurotium amstelodami Neosartorya fischeri Metarhizium anisopliae A. niger A. niveus A. oryzae A. ochraceus A. parasiticus A. puniceus 𝐴. 𝑟𝑒𝑠𝑡𝑟𝑖𝑐𝑡𝑢𝑠 𝐴. 𝑠𝑐𝑙𝑒𝑟𝑜𝑡𝑖𝑜𝑟𝑢𝑚 𝐴. 𝑗𝑎𝑛𝑢𝑠 A. clavatus 𝐴. 𝑒𝑙𝑜𝑛𝑔𝑎𝑡𝑢𝑠 A. brunneouniseriatus 𝐴. 𝑐𝑎𝑚𝑝𝑒𝑠𝑡𝑟𝑖𝑠 𝐴. 𝑎𝑒𝑛𝑒𝑢𝑠 𝐴. 𝑎𝑟𝑒𝑛𝑎𝑟𝑖𝑢𝑠 a b NT: nontested. Type strains designated by a superscript T. 4 BioMed Research International

Table 2: Phenotypic characters of Aspergillus species originating from Greece.

Conidia Species ATHUM number Vesicle shape Vesicles (𝜇m) Metulae (𝜇m) Phialides (𝜇m) Size (𝜇m) Ornamentation Aspergillus aculeatus 5028 Globose 36–50 — 6–10 × 3.5–5 4-5 Finely rough Aspergillus clavatus 5032, 5036 Clavate 15–150 × 15–55 — 7–10 × 2.5–4 4-5(-8) × 2.8–4 Smooth Aspergillus flavus 5015 Globose 30–55 9–13 × 4-5 9–14 × 2-3 3–6 Smooth Aspergillus fumigatus 5013 Spathulate (13-)17–22 — 6–9 × 2-3 2.2–3.5 Minutely echinulate Aspergillus niger 2539, 5044 Globose 40–65 14–22 × 4–6 7–9 × 3-4 4–5.5 Rough Aspergillus niveus 5029 Spathulate 9–16 6-7 × 2-3 6-7 × 2-3 2-3 Smooth Aspergillus ochraceus 5014 Globose 22–40 10–15 × 4-5 10-11 × 2-3 2-3 Smooth Aspergillus oryzae 4958 Subglobose 25–45 9–12 × 4-5 9–12 × 4–6 5–8 Rough Aspergillus parasiticus 5037, 5038 Globose 25–40 — 7–11 × 3-4 3.5–6 Rough Aspergillus sydowii 5093 Pyriform 8–17 4–6(-7) × 3-4 6–8 × 2-3 3-4 Echinulate Aspergillus terreus 4761 Globose 10–17 5-6 × 2-3 7-8 × 1.5–2 2–2.5 Smooth Aspergillus ustus 5097, 5103 Pyriform 11–17 5–7 × 3.5–4 4–7 × 2.5–3.5 3-4 Rough Aspergillus versicolor 2541 Pyriform 10–15 × 3–5 5–7 × 3–5 5–8 × 3–5 3-4 Rough Emericella nidulans 5022 Spathulate 10–14 7-8 × 3-4 6-7 × 2-3 3-4 Rough Emericella variecolor 5019 Spathulate 16–19 6–9 × 2-3 6–9 × 2-3 2-3 Rough Eurotium amstelodami 5082 Subglobose 15–35 — 6–9 × 3-4 4–6 Echinulate Neosartorya fischeri 5030 Spathulate 12–16 — 6-7 × 2–2.5 2-3 Smooth

analyses were identical (data not shown). All sequences of and 805–847 for cox1), reflecting to the apparent genetic Aspergillus species and their teleomorphs not amplified in variability between different Aspergillus species. Amplicons this study were taken from GenBank and are provided with were sequenced in both directions, sequences were combined their Accession Numbers and the species names provided and compared with all known related sequences in the data- bythedatabase.AlignedmatricesareavailableinTreebase, banks, and appropriate phylogenetic trees were constructed. http://purl.org/phylo/treebase/phylows/study/12165. Phylogenetic trees deduced from NJ analyses were drawn (Figures 2, 3, 4, 5,and6), and parsimony and Bayesian methods were used to examine the sensitivity of the resulting 3. Results trees and tree topologies. Trees remained largely invariant to these manipulations, and topologies were similar for 3.1. Morphological Analysis. The cultural characteristics of each gene tested independently of the phylogenetic method the Aspergillus spp. have been recorded for all the strains applied. and their evaluation was critical in cases of closely related The phylogenetic analysis of ITS and benAsequences species. The colony texture, colour and growth rate onthe included 227 and 257 taxa, respectively, belonging to 210 nutrient media, the formation of exudates, soluble pigments and 235 species of Aspergillus and their teleomorphs (i.e., and sclerotia, and the thermotolerance were examined. The Emericella, Eurotium, Fenellia, Neocarpenteles, Neosartorya, most important diagnostic characters are the morphological Neopetromyces, Petromyces, and Warcupiella), in order to features of the conidial heads, including their growth pattern, determine their phylogenetic positions (Figures 2 and 3). For size and seriation, dimensions and surface texture of the the datasets of IGS (Supplemental Figure S1 of Supplementary conidiophore, the shape and size of the metulae, phialides Material available online at http://dx.doi.org/10.1155/2013/ and conidia, and the colour and the wall ornamentation of 260395)andcox1 (Supplemental Figure S2) the sequences the phialidospores (Figure 1). The range of variability found analysed were significantly fewer than above, since there are for the main morphological characteristics of the strains no other entries in the databanks apart from those examined of Aspergillus spp. originating from Greece is presented in in our work (Table 1). Finally, the datasets of rpb2(Figure 4) Table 2. In addition, the morphology of the teleomorph and rns (Figure 5) were enriched with the addition of 147 and has been examined in detail when the holomorph was 10 more species, respectively, from databanks. present. There is a great morphological variation in the type The ITS dataset included 634 characters, 376 of which of ascomata, from loosely interwoven hyphae to compact were parsimony informative, resulting to 10 parsimonious sclerotioid structures, as well as in the size, ornamentation, trees (tree length: 2,606, consistency index (CI): 0.360 and and sculpture of the ascospores. retention index (RI): 0.824). Among the 790 polymorphic sites of the benA sequences, 533 were phylogenetically infor- 3.2. DNA Analyses. Amplified parts of genes and regions mative.ThetopologyoftheNJtreeisthesameasoneof had variable sizes (i.e., in bp, 478–532 for ITS, 650–850 for the 9,980 most parsimonious trees inferred by the PAUP IGS, 411–587 for benA, 785–811 for rpb2, 536–555 for rns programme (length: 7,986 steps, CI: 0.183, and RI: 0.754). The BioMed Research International 5

(a) (b) (c)

(d) (e) (f)

(g) (h) (i) (j)

(k) (l) (m) (n)

Figure 1: Anamorphic and teleomorphic characters of Aspergillus spp. (a) Aspergillus ochraceus ATHUM 5014. (b) Aspergillus flavus ATHUM 5033. (c) Aspergillus parasiticus ATHUM 5037. (d) Aspergillus fumigatus ATHUM 5013. (e) Aspergillus clavatus ATHUM 5036. (f) Aspergillus giganteus NRRL 10. (g) Aspergillus janus NRRL 1787. (h) Aspergillus versicolor ATHUM 2541. (i) Aspergillus sydowii ATHUM 5093. ((j)– (k)) Eurotium amstelodami ATHUM 5082. ((l)–(n)) Neosartorya fischeri ATHUM 5030. ((a)–(i), (k), (n)) Conidial heads with phialides and conidia. (j) Part of cleistothecium with asci and ascospores. (l) Cleistothecia. (m) Ascospores. Bars = ((a)–(e), (g)–(n)) 10 𝜇m; (f) 20 𝜇m; (l) 50 𝜇m.

rpb2andcox1 dataset included 1,140 and 908 characters, with CI:0.354,andRI:0.516).Finally,therns dataset included 614 655 and 303 parsimony informative characters, and produced characters, with 130 parsimony informative characters (tree 20 and 1,185 equally parsimonious trees (tree length: 8,927 length: 512, CI: 0.534, and RI: 0.731). and 1,185, CI: 0.152 and 0.594, and RI: 0.690 and 0.670), Thirty-six strains studied in this work belong to 31 respectively. The IGS dataset included 901 characters, with Aspergillus species (see Table 1). ITS sequences from these 735 parsimony informative characters (tree length: 5,829, strains, together with sequences from other species of the 6 BioMed Research International

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Figure 2: Continued. BioMed Research International 7

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Figure 2: Phylogenetic tree constructed from unambiguously aligned DNA sequences of the ITS domain as produced by NJ. Sequences obtained during this study are presented in bold, whereas those retrieved from GenBank are shown in roman with their Accession Numbers. Clade credibility using NJ calculated from 1,000 replicates (numbers in roman), parsimonial BS support calculated from 100 replicates (numbers in italics) using PAUP, and PPs produced by 1,000,000 generations (numbers in bold) using MrBayes are shown. 8 BioMed Research International

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Figure 3: Continued. BioMed Research International 9

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Figure 3: Phylogenetic tree constructed from unambiguously aligned combined DNA sequences of the benA gene as produced by NJ. Sequences obtained during this study are presented in bold, whereas those retrieved from GenBank are shown in roman with their Accession Numbers. Clade credibility using NJ calculated from 1,000 replicates (numbers in roman), parsimonial BS support calculated from 100 replicates (numbers in italics) using PAUP, and PPs produced by 1,000,000 generations (numbers in bold) using MrBayes are shown. 10 BioMed Research International

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Figure 4: Continued. BioMed Research International 11

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Figure 4: Phylogenetic tree constructed from unambiguously aligned combined DNA sequences of the nuclear rpb2 gene as produced by NJ. Sequences obtained during this study are presented in bold, whereas those retrieved from GenBank are shown in roman with their Accession Numbers. Clade credibility using NJ calculated from 1000 replicates (numbers in roman), parsimonial BS support calculated from 100 replicates (numbers in italics) using PAUP, and PPs produced by 1,000,000 generations (numbers in bold) using MrBayes are shown.

genus that were retrieved from the GenBank (altogether ranged from 51–100%. Members of Candidi, Cervini, and around 200), clustered well within their corresponding sec- Circumdati clustered with 100% and likewise Raperi, and tions of genus Aspergillus (Figure 2). The NJ, MP bootstrap, Restricti showed higher than 90% support for all three meth- and Posterior Probabilities (PPs) support of all sections ods. Species of sections Aspergillus (Eurotium xerophilum and 12 BioMed Research International

Figure 5: Phylogenetic tree constructed from unambiguously aligned combined DNA sequences of the mitochondrial rns gene as produced by NJ. Sequences obtained during this study are presented in bold, whereas those retrieved from GenBank are shown in roman with their Accession Numbers. Clade credibility using NJ calculated from 1,000 replicates (numbers in roman), parsimonial BS support calculated from 100 replicates (numbers in italics) using PAUP, and PPs produced by 1,000,000 generations (numbers in bold) using MrBayes are shown.

Eurotium halophilicum are excluded) and Flavi (A. alliaceus but sections Bispori, Ochraceorosei, and Sparsi are scattered. and A. leporis are excluded) presented higher than 90% sup- Finally, strains from different sections, that is, A. bisporus portforatleasttwodifferentapproachesofbootstrapanal- NRRL 3693 (Bispori), A. brunneouniseriatus NRRL 4273 (Cre- yses, whereas sections Clavati, Cremei, Fumigati, and Sparsi mei), A. aeneus NRRL 4769 (Nidulantes)andA. paradoxus clustered with excellent support (>90%) for one methodology AY373860 (Clavati)formedamixedcluster(Figure 2). This and at least good (51–89%) for the other two (Figure 2). cluster grouped as a sister clade to a group consisting of A. Species belonging to sections Nigri, Nidulantes, and Usti crystallinus and A. malodoratus (see Figure 2)withexcellent were divided into two well discriminated, well supported NJ, MP and PP (100%) support. groups in each case (Figure 2). Subgenus Nidulantes based All benA amplicons contained three introns with the on this dataset seems to be polyphyletic; that is, sections exception of Emericella variecolor and A. versicolor which Nidulantes, Raperi, Silvati, and Usti form a single cluster, lackthelastintron.AsshowninFigure 3,thesingletree BioMed Research International 13

Figure 6: Phylogenetic tree constructed from unambiguously aligned concatenated DNA sequences of the nuclear ITS, IGS, benA, and rpb2 genes and the mitochondrial cox1andrns genes as produced by NJ. Sequences obtained during this study are presented in bold, whereas those retrieved from GenBank are shown in roman with their Accession Numbers. Clade credibility using NJ calculated from 1,000 replicates (numbers in roman), parsimonial BS support calculated from 100 replicates (numbers in italics) using PAUP, and PPs produced by 1,000,000 generations (numbers in bold) using MrBayes are shown. Solid lines with numbers in circles represent the seven clusters, while the dotted lines show the sections of the species examined and their teleomorphs.

based on benA nucleotide sequences discriminated clearly A. bahamensis, A. homomorphus, and A. uvarum; Figure 3). species that belong to sections Candidi, Cervini, Circumdati, Species within sections Aspergillus, Cremei, Nidulantes, Peni- Clavati, Flavi, Fumigati, Nigri, Raperi and Terrei.Topology cillium, Usti, and Restricti although they showed good cluster- support was extremely good for these sections with at least ing, they failed to produce high bootstrap support due to the one methodology showing 100% bootstrap value in all cases odd discrepancies and must therefore be approached more (except section Sparsi:98,92,and62%forNJ,MPbootstrap cautiously. and PP, resp.). As in the case of the ITS dataset section In accordance with ITS and benA, analyses of the rpb2 Nigri was divided into two groups with the second group dataset provided similar or better phylogeny (Figure 4)for containing five members (i.e., A. aculeatus, A. aculeatinus, species of sections Aspergillus, Clavati, Cervini, Circumdati, 14 BioMed Research International

Fumigati, Penicillium, Raperi, and Restricti grouping with be related to the developmental patterns of the teleomorphs excellent bootstrap support (93–100%, irrelevant to the boot- (Figure 6). In the subgenus Circumdati, clade 1 includes mem- strap methodology applied). Here again, section Nigri species bers of section Circumdati with connection to Neopetromyces were placed into two distinct groups with bootstrap support [40], a genus with light-coloured multilocular ascostromata. of 100, 100, 99% and 77, 56, 98% (for the NJ and MP bootstrap It is distantly related to section Flavi (clade 6), with a recently and PP of the BI analyses of groups 1 and 2, resp.), but the two confirmed connection to Petromyces [41, 42], characterized groups were sister clades, supported with 69, 56, and 89% NJ by black multilocular ascostromata and section Nigri (clade and MP bootstrap and PP values, with the exception of A. 7) with unknown teleomorphs, even though sclerotia are japonicus, which was placed basally to rest of the Nigri species. formed that should be considered as ascomatal primordia The phylogenies found after the analyses of IGS (Supple- [43]. In the subgenus Nidulantes,thesectionsNidulantes, mental Figure S1) and cox1 (Supplemental Figure S2) did not and Usti in clade 2 cluster with Emericella teleomorphs improve the picture drawn from ITS, benA, and rpb2treesbut that produce ascomata surrounded by Hulle¨ cells. Addi- instead added some ambiguities which could be attributed tionally, few species belonging to subgenus Nidulantes are mainly to the limited number of species examined since this in clade 3 which forms a cluster intermixed with subgenus may inhibit the good resolution of the species phylogenetic Aspergillus sections Aspergillus and Restricti that contain relationships. Nevertheless, phylogenies from both IGS and Eurotium teleomorphs with thin layered ascomata loosely cox1 can be used for better distinguishing species within the suspended in the mycelium. In the subgenus Fumigati,in same section (e.g., A. niger with A. awamori), as also found clade 5, the section Fumigati is connected to Neosartorya in a recent paper with other genes by Perrone et al. [39], with ascomata composed of abundant sterile hyphae and the but are not suitable to discriminate the sections themselves. section Clavati is connected to Neocarpentelles with uniloc- Finally,thetreeproducedbythemitochondrialrns dataset ular stromatic ascomata. Clade 4 includes taxa in sections showed very good clustering of species belonging to sections Terrei, Candidi, and Bispori without evident teleomorphic Aspergillus, Clavati, Circumdati, Nigri, Nidulantes, and Usti connections. (with the exception of A. elongatus which is basal to the other Usti species) with excellent bootstrap support (values range from 93 to 100%; Figure 5) and secondarily weaker bootstrap 4. Discussion values of 55–90% for species that belong to Flavi, Terrei, and Fumigati (Figure 5). Thus, a general conclusion from Fungal phylogeny based solely on morphological criteria or single gene analyses is that subgenera Aspergillus, Candidi, on single genes like the rRNA gene sequences may not always Fumigati, and Terrei evidentlyformwellsupportedclusters, elucidate the taxonomic status of the organisms examined while sections of the subgenera Circumdati, and Nidulantes [14]. Also, studies based on a single gene do not always faith- are dispersed. fullyrepresentthehistoryoftheentiregenomecontainingit To achieve full exploitation of the information obtained and comparisons may give the wrong conclusions about the from nuclear and mitochondrial genes, a tree was constructed relationship of a fungus with members of the same species or based on the combined gene datasets. The concatenated eventhesamegenus[44]. Certainly, the nuclear rDNA repeat dataset included 4,507 characters, with 2,217 parsimony is the most popular region in molecular phylogenetic studies informative characters and parsimony analysis provided a because it is multicopied and contains the highly conserved singletree.Thetreelengthwasbasedon13,497steps(CI: genes 18S, 5.8S, and 28S, as well as the variable domains 0.40, HI: 0.60, RI: 0.52, RC: 0.21). Analysis of the same of ITS1 and ITS2, and the nontranscribed IGS region. In dataset with NJ and BI methods produced similar trees with addition, the 18S and 28S regions often harbour group I identical topologies wherever there was a strong support introns, inserted at highly conserved positions, contributing (Figure 6). The sections that contained the larger numbers further information in regard to this region. These properties of strains examined, that is, Flavi, Nigri, Nidulantes,along have been exploited at length in studies of Aspergillus species with sections Aspergillus, Circumdati, Clavati, Fumigati, and [11, 15, 45]orotherfungalgenera[28, 46, 47]. In a similar Terrei, were well discriminated with better support compared way, the mtDNA of fungi has lately attracted considerable to the results produced by single gene analyses (Figure 6). interest as an alternative or complementary molecule for In addition, the tree produced by the concatenated dataset phylogenetic studies [26, 35, 48], because of its high copy further supports the polyphyly of subgenus Circumdati,as numbers, richness in AT sequences, lack of methylation, single gene phylogenies have also shown, since sections Nigri, universal gene functions, highly conserved regions, as well Flavi, and Circumdati form separate clades at the base of as variable domains and introns [49]. Currently, the most the tree. The section Circumdati,representedinthisstudy common mt genes used in fungal phylogenetic studies are by species A. ochraceus and A. sclerotiorum,formsasister rns and cox1, because the primers designed for these genes clade to section Zonati,representedbyA. clavatoflavus. Both can be applied to a wide range of taxonomically different sections can be a sibling clade to section Nidulantes based fungi [50, 51]. Our analyses of representative species from the only on the NJ analysis. Both MP and BI analyses show that most common sections of Aspergillus showed for the first time this relation is not supported and thus sections Circumdati here that mitochondrial based phylogenies indicate different and Zonati collapse at the base of the tree. evolutionary pathways which may provide valuable data for Most importantly, the tree of the concatenated dataset taxonomic purposes when combined with morphological presented a backbone of seven major clades which could and nuclear based molecular datasets. BioMed Research International 15

Ithasoftenbeenarguedthatthereconstructionof suggestthatsectionUsti should be retained as an independent phylogenies is better achieved by multiple datasets, not only section within the subgenus Nidulantes with further support, of nuclear but also of mitochondrial origin [52]. In the present since both the ITS and concatenated data analyses show that study, the ITS and IGS regions from the nu-rRNA gene species of Usti, Nidulantes, Silvati, and Raperi comprise a complex and rns and cox1 from the mt genomes were chosen single clade supported (e.g., from ITS dataset) by NJ-MP to study the phylogenetic relationships of Aspergillus species. bootstrap (51% and 71%, resp.), and BI Posterior Probability To overcome possible ambiguities raised by the analyses of (95%), and thus, altogether may be considered as one group these genes, data from the nuclear genes rpb2andbenA within subgenus Nidulantes (Figures 2 and 6). that are generally accepted as phylogenetically informative Until now, several genes have been proposed for the were also used [5, 45, 53]. Although benAsequenceswere identification of species within the section of black aspergilli. excluded from the concatenated data used in the most recent However, the results obtained are very variable. IGS and multigene approach, since the presence of two or three cox1 sequences were found unsuitable to discriminate species introns in the corresponding benA amplicons was attributed within this section (Supplemental Figures S1 and S2), because to amplification of paralogous genes [23], we have chosen to they either exhibited too high intraspecies variability or this use this data in our work for three reasons: (a) because 34 variability was also extended to inter-species comparisons out of the 36 species examined contained all three introns, [3, 20]. Similarly, ITS sequences could distinguish four only two species were missing one intron, (b) the identity groups within the A. niger species complex but failed to levels of the 34 amplicon sequences (i.e., containing all three do so for species like A. carbonarius and A. sclerotioniger, introns) was >84%, and (c) exon identity between all 36 or A. japonicus, A. aculeatus and A. uvarum which had amplicons ranged between 86 and 96%. As shown in results, identical ITS sequences [20]. Also, mt cytochrome b (cob) this choice was justified because the benA based phylogenetic gene sequences were informative for many species within tree provided additional information as, for example, in the Nigri section but they failed to distinguish between A. the case of members of section Nigri that were clustered niger and A. tubingensis [54]. Thus, the best discriminatory together but clearly differentiated the uniseriate from the results based on single gene sequences were those obtained biseriatespecies.Thus,alldatafromnuclearandmtgenes from 𝛽-tubulin and calmodulin phylogenetic analysis which were combined as a single unit in order to blend information placed 26 taxa within subgenus Circumdati section Nigri, from two independent heritage lines, to examine whether this further dividing them into 5 main clades, grouping all approach provides useful information in resolving phyloge- uniseriate species (7) in only one clade (the A. aculeatus netic ambiguities within the genus Aspergillus. clade) [13, 45].Inthepresentwork,singlegenedatasetswith The single gene trees obtained from ITS, IGS, rpb2, and many individual sequences (ITS, benA, and rpb2) and the rns and, mainly, the concatenated datasets, strengthen the concatenated dataset clearly distinguished members of Nigri fraternisation of Clavati to subgenus Fumigati,asspeciesof and divided them into two groups: the first containing A. section Clavati form a sister group to species within section aculeatus, A. aculeatinus, A. bahamensis, A. uvarum—in all Fumigati,withMPbootstrapandPPsupportthatreached89 datasets—and A. homomorphus, A. japonicus—wherever data and 100%, respectively, at the combined dataset (Figure 6). wereavailable—,andthesecondcomposedofalltherest This is in full accordance with results obtained from a recent species (see e.g., ITS or benA trees, Figures 2 and 3). It is multilocus analysis that was based solely on nuclear genes interesting to note that these two clades correspond to the and the addition of section Cervini species in this subgenus uniseriate and biseriate conidial head formation, which may [23, 24]. Interestingly enough, our analysis based on the be a possible explanation why previous studies that did not ITS dataset showed that although sufficient bootstrap or take into account this characteristic, failed to cluster them posterior probability support was lacking, uniseriate species accordingly [13, 20, 45]. An additional explanation for the that belong to subgenera Fumigati and Aspergillus clustered splitting of section Nigri species into two subgroups may be together, and apart from other species of Aspergillus that are attributed to the range of variability resulting from adaptation biseriate (Figure 2). of the species to the Mediterranean type ecosystem. This is Section Usti is of particular interest since its elimination also evident by the variability found in the morphological and the transfer of species A. ustus, A. deflectus, A. puniceus, characteristics of the Greek Aspergillus isolates (Table 2). A. granulosus, and A. pseudodeflectus to section Nidulantes, The biseriate species within section Nigri cluster with good and species A. conjunctus, A. funiculosus, A. silvaticus, A. bootstrap support (67% bootstrap for NJ and 84% PP) with panamensis and A. anthodesmis to section Sparsi was initially biseriatespeciesbelongingtosectionsFlavipedes, Terrei, proposed [15], only to be rejected a few years later, by Ochraceorosei, and Sparsi,whiletheuniseriatespeciesof exploiting the results of polyphasic approaches in which the Nigri are basal to the above-mentioned biseriate clades of sequences of the nuclear 𝛽-tubulin, calmodulin, actin, and sections Flavi with good support (70% PP, Figure 2). Thus, ITS genes were used [5, 18]. These works not only preserved the differentiation of Nigri species studied here into uniseriate section Usti but also added eight more species in it, namely, and biseriate not only helped to group them in combination A. ustus, A. puniceus, A. granulosus, A. pseudodeflectus, A. with molecular markers into two clearly distinct clades, but calidoustus, A. insuetus, A. kevei, and Emericella heterothal- in addition clustered together biseriate species of all sections. lica. Finally, more recent works added nine more Aspergillus Species of the Flavi section were grouped well together, species in it, seven and two, respectively, bringing the total with high bootstrap support (>80%), irrelevant to the dataset number to 21 [11, 24]. In full agreement with these, our results used. While single gene analysis, like benA, failed to separate 16 BioMed Research International

A. flavus from A. oryzae, the concatenated dataset clearly In conclusion, our study clearly shows that all molecules demonstrated its advantages. It could fully discriminate all used contributed to the resolution of phylogenetic relation- species examined and could also confirm that taxa within ships in Aspergillus, irrespective to the different ways that the section, like A. oryzae, A. flavus, and A. parasiticus thephylogeneticdatawereprocessed.Italsoconfirmsthat arephylogeneticallydistantlyrelatedtoA. ochraceus,as single gene based analyses do not solve all ambiguities and previously proposed [55]andalsorecentlyshownwiththe do not always represent the evolutionary history of the polyphyly of A. flavus, and in extent of section Flavi [56]. species. Certainly, Aspergillus is a very diverse genus and it Previous reports showed that sections Circumdati, Flavi is not always easy to determine the phylogenetic relationships and Nigri do not cluster together [23] and this was further between species. Thus, the combination of sequence informa- confirmed by our phylogenetic analyses of ITS, benA, and tion from nuclear and mitochondrial genes resolves several rpb2 datasets. However, it must be pointed out that this is circumscriptions and relationships of Aspergillus species in contradiction with our morphological observations which within different sections and demonstrates the advantages of this multigenic approach. It is expected that the vast amount demonstrated significant morphological similarities, that is, of information derived from the released complete genomes biseriateconidialheads,largeglobosevesicles,ascostromata of Aspergillus specieswillbeutilizedinfuturestudiesbymany when teleomorph present, and suggest a possible common researchers to select new additional genes from both (nuclear origin of these sections. The latter is further supported andmitochondrial)evolutionarylines.Suchcombineddatais by the analysis of the mt rns sequences, even though the expected to provide the appropriate levels of resolving power bootstrap values do not seem significant enough. Thus, in future phylogenetic studies. this intriguing controversy between molecular data and morphological observations underlines the necessity to use polyphasic approaches in one hand and increase the number Acknowledgments of species examined on the other in order to solve difficult The authors are greatly indebted to Dr. Stephen Peterson, taxonomy problems. Peoria, USA, for providing ex-type strains of Aspergillus spp. The single tree produced by our concatenated dataset This research was supported by the Greek General Secretariat showed that although A. niveus and A. terreus,whichbelong of Research and Technology (Project TR-5) in the frame of the to section Terrei, are closely related and with excellent support “Competitiveness” Program of European Commission. (100% irrelevant of the method applied), they are far apart from A. janus (section Flavipedes)andA. arenarius,both ofwhichwerepreviouslyplacedcloselytospeciesofTerrei, References Flavipedes, and Candidi [23]. 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